Ouabain

Reagents for ACSF and internal solutions, biocytin, NBQX, BaCl₂, and picrotoxin were obtained from Sigma-Aldrich. CNQX, AP-5, DHK, DL-TBOA, TTX, ouabain, and D-serine were obtained from Tocris. L-glutamic acid from BioTrend and SR-101 from Invitrogen. NBQX, CNQX, and DL-TBOA were dissolved in DMSO. picrotoxin was dissolved in EtOH. AP-5, D-serine, TTX, ouabain, and DHK were dissolved in ddH₂O.
In both patch-clamp and two-photon imaging experiments, following baseline recordings, drugs were applied in the external solution for at least 15 min prior to recordings. For the double pharmacology imaging experiments (Fig. 9) following baseline recordings, DHK (300 μM) was first applied and recordings were acquired 20 min later. Subsequently, DHK (300 μM) and DL-TBOA (68 μM) were applied for 20 min before recording. In a number of recordings in which we applied much higher doses of DL-TBOA (300 µM), we observed a change in baseline iGluSnFr fluorescence (similar to ref. ^{18 (link)}), a reduced amplitude of the evoked responses and cellular swelling often accompanied by a lateral or Z drift. These experiments had to be excluded, which explains the low n value for this set of experiments (Supplementary Fig. 9).

HAC15 cells were provided by Professor WE Rainey (University of Michigan, Ann Arbor, MI, USA). Cells were cultured in DMEM/F12 with 10% Cosmic Calf serum (HyClone, Logan, UT, USA); detailed methods of cell culture are described in our previous report [59 (link),62 (link)]. Ouabain was obtained from Tocris Bioscience (#1076, Ellisville, MO, USA).